Antimicrobial peptides have been isolated from a wide variety of animal sources. These sources include, prominently, leukocytes of humans (Lehrer, R. I. et al., Ann Rev Immunol (1992) 11:105); pigs (Kokryakov, V. N. et al., FEBS Lett (1993) 231); bovine sources (Selsted, M. E. et al., J Biol Chem (1993) 268:6641); rabbits (Patterson-Delafield, J. et al., Infect Immun (1980) 30:180); and birds (Harwig, S. S. L. et al., FEBS Lett (1994) 342:281). Antimicrobial peptides have also been found in bovine tongue (Schonwetter, B. S. et al., Science (1995) 267:1645) respiratory tract epithelia (Diamond, G. et al., Proc Natl Acad Sci USA (1991) 88:3952) and gastrointestinal and genital urinary tracts of humans and animals (Jones, D. E. et al., J Biol Chem (1992) 267:23216; Bensch, K. W. et: al., FEBS Lett (1995) 368:331). In addition, antimicrobial peptides have been isolated from the hemocytes of the Horseshoe Crab as described by Nakamura, T. et al., J Biol Chem (1988) 263:16709-16713. These various antimicrobial peptides, for example the tachyplesins, polyphemusins, defensins, clavanins and gallinacins, are typically characterized by specific positions of cysteine residues which putatively control conformation of the molecule.
An additional class of antimicrobial peptides, found in the skin of the African clawed frog, Xenopus laevis, are .alpha.-helical (noncovalent-cyclic) peptides (Zasloff, M., Proc Natl Acad Sci USA (1987) 84:5449). This class of antimicrobial peptides, called the magainins, in their mature form contain, 23 amino acids and are .alpha.-helical but not amidated. The magainins possess broad spectrum antimicrobial activity (Harwig, S. S. L. et al., FEBS Lett (1994) 342:281; Zasloff, M. et al., Proc Natl Acad Sci USA (1988) 85:910). The nature of the antimicrobial activity as related to the .alpha.-helical amphipathic structure of magainins has been studied (Duclohier, H. et al., Biophys J (1989) 56:1017) as has that of another class of .alpha.-helical antimicrobial peptides, the cecropins (Christensen, B. et al., Proc Natl Acad Sci USA (1988) 85:5072. The magainins are synthesized from a large prepropeptide containing a single copy of Magainin-1 and five copies of the closely related Magainin-2 (Terry, A. S. et al., J Biol Chem (1988) 263:5745).
Antimicrobial peptides and proteins have also been found in plants as reviewed by Cornelissen, B. J. C. et al., Plant Physiol (1993) 101:709-712.
The present invention is directed to a class of peptides and peptide-like compounds several members of which may be isolated from the hemocytes of the tunicate Styela clava. Tunicates are simple marine invertebrates whose larval forms contain a constellation of features establishing their kinship to early vertebrates. The body cavity of the mature tunicate provides an acceptable source of mesoderm-derived phagocytes (hemocytes) that are counterparts to the blood leukocytes of higher vertebrates. It is known that phagocytes of freshly harvested colonial tunicates are often filled with various bacteria and that the introduction of bacteria beneath the tunic is capable of inducing phagocytic cells to traverse the underlying epithelium and surround these foreign objects.
Co-pending Application Attorney Docket number 22000-20563.00 filed Nov. 6, 1996 describes a class of peptides isolated from Styela clava which are designated the clavanins. These peptides have a distinctive pattern of basic and hydrophobic amino acids side-chains. The present invention relates to another class of peptides also obtainable from these organisms which have a different pattern of amino acid sequence but also are antimicrobial. These peptides are therefore designated "clavaspirins."